Limonene Synthase Mutants Reveals Determinants of Catalytic Outcome in a Model Monoterpene Synthase

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Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

Narayanan Srividya, Edward M. Davis, Rodney B. Croteau1, and B. Markus Lange1

Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163

Contributed by Rodney B. Croteau, February 3, 2015 (sent for review November 8, 2014; reviewed by David E. Cane and Philip J. Proteau)

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of function (3). The active site harbors a highly conserved, L-aspar- spearmint (Mentha spicata L.) were used to infer which amino acid tate-rich, DDxxD motif, which is also found in prenyl elongases residues are in close proximity to the substrate and carbocation (5) and a less conserved NSE/DTE motif. The L-aspartate resi- + intermediates of the enzymatic reaction. Alanine-scanning muta- dues bind a trinuclear cluster of divalent metal ions (Mg2 or + genesis of 48 amino acids combined with enzyme fidelity analysis Mn2 ) involved in the binding and activation of the diphosphate [percentage of (−)-limonene produced] indicated which residues moiety, thereby generating characteristic carbocation intermediates are most likely to constitute the active site. Mutation of residues (Fig. 1). Enzymes catalyzing these ionization-initiated cyclization W324 and H579 caused a significant drop in enzyme activity and reactions are commonly referred to as class I TPSs (as opposed to formation of products (myrcene, linalool, and terpineol) character- class II TPSs that initiate cyclizations by protonation) (3). The istic of a premature termination of the reaction. A double mutant remaining course of MTS catalysis is variable and generates (W324A/H579A) had no detectable enzyme activity, indicating that acyclic, monocyclic, and/or bicyclic monoterpenes. either substrate binding or the terminating reaction was impaired. Because of the widespread occurrence of (−)-(4S)-limonene Exchanges to other aromatic residues (W324H, W324F, W324Y, throughout the plant kingdom, the relative simplicity of catalysis, H579F, H579Y, and H579W) resulted in enzyme catalysts with sig- the comparatively well-understood mechanism, and the avail-

nificantly reduced activity. Sequence comparisons across the ability of a crystal structure at 2.7-Å resolution (6), (4S)-limo- BIOCHEMISTRY angiosperm lineage provided evidence that W324 is a conserved nene synthase [(4S)-LS] has become a model for understanding residue, whereas the position equivalent to H579 is occupied by catalysis by class I TPSs. The (4S)-LS gene is translated into aromatic residues (H, F, or Y). These results are consistent with a crit- a preprotein with an N-terminal targeting sequence for trans- ical role of W324 and H579 in the stabilization of carbocation inter- port to the plastidial envelope membrane (7). Based on results mediates. The potential of these residues to serve as the catalytic obtained with a series of truncated (4S)-LS mutants, the most base facilitating the terminal deprotonation reaction is discussed. likely cleavage site of the preprotein was determined to be at (or near) a tandem pair of arginines (R58 and R59 of the preprotein) monoterpene synthase | enzyme catalysis | mechanism | carbocation | (8). If truncated beyond R58, (4S)-LS shows no cyclization ac- structure–function relationship tivity with GPP but is fully functional with linalyl diphosphate (LPP) as a substrate. Interestingly, the crystal structure of (4S)-LS, erpenoids are a structurally diverse group of metabolites with complexed with the nonhydrolyzable LPP analog 2-fluorolinalyl Tfunctions in both primary and secondary (or specialized) diphosphate (FLPP), does not provide evidence for a direct metabolism. Primary metabolites derived from terpenoid path- interaction of R58 or R59 with the substrate (9). However, weak way intermediates in plants include sterols, carotenoids, and the side chains of chlorophylls, tocopherols, and quinones of elec- Significance tron transport systems. Many plant hormones are also products of terpenoid metabolism, including abscisic acid, cytokinins, brassi- Terpene synthases catalyze complex, chain length-specific, elec- nosteroids, and strigolactones (1). Secondary plant metabolites of trophilic cyclization reactions that constitute the first committed terpenoid origin can play critical defense-related roles (e.g., ses- step in the biosynthesis of structurally diverse terpenoids. quiterpene lactones and triterpene saponins serve as antifeedants) (4S)-limonene synthase [(4S)-LS] has emerged as a model en- and are dominant constituents of essential oils and resins (mono-, zyme for enhancing our comprehension of the reaction cycle

sesqui-, and diterpenes) (2). Terpene synthases (TPSs) convert of monoterpene (C10) synthases. While the stereochemistry a prenyl diphosphate of a specific chain length to the first path- of the cyclization of geranyl diphosphate to (−)-(4S)-limonene way-specific (often cyclic) intermediate in the biosynthesis of each has been the subject of several mechanistic studies, the struc- class of terpenoids. Whereas some terpene synthases are re- tural basis for the stabilization of carbocation intermediates and markably specific and only generate one product from a prenyl the termination of the reaction sequence have remained enig- diphosphate precursor, others release a larger number of products matic. We present extensive experimental evidence that the ar- from a common substrate, thus contributing to terpenoid chem- omatic amino acids W324 and H579 play critical roles in the ical diversity (3). The genomes of plants may only contain one stabilization of intermediate carbocations. A possible function of TPS gene [e.g., ent-kaurene (diterpene) synthase in the moss these residues as the terminal catalytic base is also discussed. Physcomitrella patens (Hedw.) Bruch & Schimp.], but often harbor sizable families of TPS genes with more than 20 mem- Author contributions: N.S., E.M.D., R.B.C., and B.M.L. designed research; N.S. and E.M.D. performed research; N.S., R.B.C., and B.M.L. analyzed data; and N.S. and B.M.L. wrote the bers, which is another source of terpenoid structural variety (4). paper. All monoterpene synthases (MTSs) use either geranyl di- Reviewers: D.E.C., Brown University; and P.J.P., Oregon State University. Z phosphate (GPP) or its 2 -isomer neryl diphosphate as sub- The authors declare no conflict of interest. strate, but the sequence conservation across species is generally 1To whom correspondence may be addressed. Email: [email protected] or lange-m@wsu. fairly low (2). However, MTSs share a common tertiary structure edu. αβ α (the so-called fold), with a C-terminal -domain containing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the active site and an N-terminal β-domain of as yet uncertain 1073/pnas.1501203112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1501203112 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 ACYCLIC ACYCLIC ALCOHOLS HYDROCARBON

BICYCLIC HYDROCARBONS

MONOCYCLIC HYDROCARBON

MONOCYCLIC ALCOHOLS BICYCLIC ETHER

Fig. 1. Proposed mechanism for (4S)-limonene synthase catalysis. OPP denotes the diphosphate moiety. The primary pathway in the wild-type enzyme leads to the formation of (−)-limonene (dark gray) and smaller amounts of bicyclic and acyclic products (light gray). Other products shown in this figure are released by mutant enzymes.

interactions, in particular electrostatic interplay of R58 with the catalytic base for this deprotonation in various prenyl di- E363 and hydrogen bonding between R59 and V357/Y435, ap- phosphate synthases and terpene synthases, but no direct evi- pear to anchor the N-terminal strand to the outside of the active dence is available to date (3, 14–17) and it seems highly unlikely site, thereby possibly supporting the closure of the active site, in the present case due to spatial considerations. Here we pres- while not interfering with the binding of the substrate and ent a comprehensive dataset to map the active site of spearmint intermediates (9). The reaction mechanism of (4S)-LS from (4S)-LS and evaluate residues with potential roles in stabilizing Mentha spicata spearmint ( L.) has been studied in some detail. carbocation intermediates of the reaction cycle. We also discuss The catalytic cascade involves the migration of the diphosphate the broader implications of our findings for understanding catal- group to C3 of the geranyl cation (from the original C1) to afford ysis and reaction termination by this fascinating class of enzymes. enzyme-bound (3S)-LPP as an intermediate (10) (Fig. 1). Fol- lowing C2–C3 rotation, the diphosphate is released again to Results – generate a linalyl cation. The proximity of the C6 C7 double L-Alanine Scanning Mutagenesis Defines Residues Required for bond to the positive charge facilitates an anti-SN′ cyclization to − S Substrate Binding and/or Catalysis. X-ray data for the crystal struc- form the ( )-(4 )-terpinyl cation (11, 12) (Fig. 1). Deprotona- ture of the pseudomature form of spearmint (4S)-LS (R58; lack- tion from the adjacent methyl group (C8 of the original GPP) ing the plastidial targeting sequence), complexed with FLPP as yields the monocyclic olefin (−)-(4S)-limonene as the major a nonhydrolyzable analog of the reaction intermediate LPP (9), product (96%). Side products are obtained by either premature deprotonation of the geranyl cation to generate the acyclic olefin were used to infer the amino acid residues within closest prox- myrcene (2%) or additional cyclization of the terpinyl cation imity to the substrate (Dataset S1). Distances were calculated (between C2 and C7 of the original GPP) to produce, after between each of the 10 carbon atoms of the substrate analog and deprotonation, a mixture of the bicyclic olefins α- and β-pinene the carbon atoms of all amino acids of the protein (except for (2%) (10, 13) (Fig. 1). those forming peptide bonds). The 48 residues located closest to Using synthetic analogs of GPP with a chiral methyl group at the substrate analog were exchanged for L-alanine by site-directed C9 (carrying 1H, 2H, and 3H), Coates et al. demonstrated that mutagenesis (introduced into the pseudomature form of (4S)-LS the final deprotonation occurs predominantly by re-facial anti- that is truncated at R58), and these mutant enzymes were ex- elimination (12). It has been hypothesized that the diphosphate pressed in Escherichia coli. Following purification over hy- anion released from the prenyl diphosphate substrate may act as droxyapatite, each recombinant enzyme was incubated with GPP

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1501203112 Srividya et al. Downloaded by guest on September 23, 2021 and products of the reaction were analyzed by quantitative chiral the prenyl diphosphate, and thus do not interact with the prenyl phase gas chromatography. or emerging terpenoid moiety (3) (Fig. 2). Mutations affecting the conserved L-aspartate residues known to be of crucial importance for complexing divalent active site W324A and H579A Mutants Are Catalytically Impaired and Preferentially + + metal ions (Mg2 or Mn2 ) in monoterpene synthases and re- Generate an Acyclic Hydrocarbon and Monoterpene Alcohols. Under lated enzymes (5, 18), D352A, D353A, and D496A, predictably routine end-point assay conditions (200 μg purified protein), all resulted in inactive enzymes (Dataset S2). The majority of the available substrate (GPP concentration at 5 mM) was turned over remaining mutants generated product profiles similar to those of by the wild-type and most mutant enzymes. However, two mutants, the wild-type enzyme [(−)-limonene (96.6%), myrcene (1.0%), W324A and H579A, converted only small amounts of substrate (−)-α-pinene (0.6%), (+)-α-pinene (0.7%), (−)-β-pinene (0.5%), during the 2-h time frame of the assay. Under kinetic assay con- ≤ (+)-β-pinene (0.5%), and (+)-sabinene (0.3%)]. However, some ditions, when the R58 wild-type enzyme converts 50% of the substrate, the specific activity of R58 was 0.19 nmol (h × mg pro- single residue exchanges led to a significantly reduced rate −1 of (−)-limonene production, with increases in the formation tein) , and the catalytic constants determined in our assays were similar to those reported in the literature (Km = 12.6 μM; kcat = of side products and novel monoterpenes not detectable in assays − 0.037 s 1) (8). The specific activities of the W324A and H579A with the wild-type enzyme (Dataset S2). The acyclic monoter- mutant enzymes were significantly reduced and, therefore, kinetic pene myrcene was produced in substantial amounts by M458A values could not be determined with accuracy. (10.1%), I348A (9.7%), and T349A (8.3%). Acyclic monoterpene To further investigate the potential roles of W324 and H579 in alcohols were released at fairly high levels from mutant enzymes (4S)-LS catalysis, additional mutants were generated. W324 and W324A [equal amounts (26.7% each) of (−)- and (+)-linalool], − + H579 were exchanged with another aromatic residue (W324H, M458A [equal amounts (14.3% each) of ( )-and ( )-linalool], W324F, W324Y, H579F, H579Y, and H579W), a base (W324H, and H579A [8.6% (−)-linalool] (Dataset S2). A novel product R W324K, and H579K), or a randomly selected amino acid. The derived from an altered reaction stereochemistry was (4 )- majority of W3234 mutant enzymes had extremely low specific + − ( )-limonene generated by M458A (8.6%). Monocyclic alcohols activities [≤0.02 nmol (h × mg protein) 1;3–10% of wild type], + α were found to be novel products of M458A [27.3% ( )- -terpineol], the only exception being W324F and W324Y [0.08 and 0.06 nmol − α α − H579A [25.0% ( )- -terpineol], and L492A [8.0% (-)- -terpineol] (h × mg protein) 1; 50% and 32% of wild-type activity, re- (Dataset S2). Increased amounts of bicyclic products were formed spectively]. The H579 mutants had specific activities ranging − + α by N345A [36.6% ( )-sabinene, 14.8% ( )- -pinene, and 13.9% between 40% and 50% of those of the wild-type enzyme. The BIOCHEMISTRY (+)-β-pinene]. Additional novel products generated in smaller product profiles were significantly altered in all 12 W324 mutants amounts by mutant enzymes were α-thujene (N345A) and (W324A, W324C, W324F, W324H, W324I, W324K, W324L, 1,8-cineole (T349A, L492A, and M458A) (Dataset S2). W324P, W324Q, W324S, W324T, and W324Y) and five of nine To further assess the observed loss in fidelity in mutant H579 mutants (H579A, H579D, H579K, H579N, and H579W) enzymes, we plotted the distance between each amino acid and (Dataset S3). The majority of W324 and H579 mutants gen- the substrate analog in the (4S)-LS crystal structure against the erated notable amounts of the acyclic hydrocarbon myrcene percentage of (−)-limonene released by the enzyme after this (Fig. 3), which is released by premature deprotonation from the residue was mutated to L-alanine (Fig. 2). Interestingly, muta- geranyl or linalyl cation (Fig. 1). The most prominent products of tions affecting the nine amino acid residues positioned in closest W324 mutants were acyclic alcohols, the formation of which is proximity to the substrate analog (i.e., those with a carbon atom caused by premature quenching of an early carbocation in- at a distance of less than 5 Å to a carbon atom of the substrate termediate (linalyl cation) by a water molecule (Fig. 1). An analog) released (−)-limonene at less than 80% of the products, example is W324P, which forms 39.6% (−)-linalool and 40.5% indicating a possible direct role in substrate (or intermediate) (+)-linalool (80.1% acyclic alcohols) (Fig. 3A). Another notable class binding and/or catalysis. The only exceptions were D352A and of products was monocyclic alcohols [(−)-α-and(+)-α-terpineol; D353A (with minimum C–C distances of 5.1 and 8.1 Å, re- detectable in 11 W324 mutants], which are generated by quenching spectively), which are known to be required for the divalent metal of the terpinyl cation by a water molecule (Fig. 1) (19). The highest ion-mediated binding of the pyrophosphate after its release from relative amounts were produced by W324A [11.6% (−)-α-terpineol and 5.7% (+)-α-terpineol; 17.3% moncyclic alcohols] (Fig. 3A). An exception was the W324H mutant which, in addition to monoterpene alcohols, produced high amounts of bicyclic monoterpenes 100 [42.0% (+)-sabinene and 7.8% (−)-sabinene]. The catalytically impaired H579 mutants, in agreement with W324 mutant data, were enepretono noitamrofenenomiL 80 L492A also characterized by the production of considerable amounts of S454A acyclic and monocyclic alcohols. A representative example is 60 I348A H579K, which released 14.9% (−)-linalool, 14.9% (+)-linalool, T349A − α + α H579A 22.1% ( )- -terpineol, and 4.1% ( )- -terpineol (56.0% alco- N345A hols) (Fig. 3B). A W324A/H579A double mutant had no de- m] 40 latotfo% W324A tectable enzyme activity. M458A

20 D496A Discussion D352A (4S)-LS Mutants with Substituted Active-Site Residues Lose Fidelity.

[s D353A 0 An analysis of the X-ray crystallographic structure of (4S)-LS (8), 0 5 10 15 20 complexed with a substrate analog, enabled us to hypothesize Closest C-C distance to substrate [Å] which amino acid residues are likely to form the active site of the enzyme. We further hypothesized that exchanges of residues involved in substrate binding and catalysis would generate mu- Fig. 2. Mutant enzymes lose fidelity when residues that likely function in substrate binding and/or catalysis are exchanged. The closest distance of tant enzymes with lower specific activity and/or result in altered a carbon atom of each amino acid in (4S)-limonene synthase is plotted product profiles. A total of 48 amino acid residues were found to against the percentage of (−)-limonene formation when the residue is ex- be arranged within a radius of about 25 Å from any carbon atom changed with L-alanine. of the C10 substrate analog. When these residues were substituted

Srividya et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 A 100 Abundance of L-histidine in TPS catalysis was already recognized by Rajao- [%] 80 narivony et al. (22) based on experiments with histidine-directed inhibitors, but the data presented here provide to our knowledge 60 the first evidence for the involvement of a specific residue (H579). 40 One would predict that, if a charge-stabilizing residue was exchanged with a nonaromatic residue, the reaction outcome would 20 reflect early termination products. Indeed, almost all W324 mutants 0 released significant amounts of acyclic myrcene and (+/−)-linalool (up to 83% of total products), which is indicative of a reaction that terminates before the cyclization to the α-terpinyl cation (Fig. 1). Interestingly, W324F and W324Y also formed mostly early termination products (77% and 81%, respectively), indicating that other aromatic amino acids could not effectively substitute for W324. H T L F P Y It is thus not surprising that the W324 residue is conserved in an- K S A I C Q W giosperm MTSs (Dataset S4). H579 mutants generally released a mixture of myrcene, (+/−)-linalool and (+/−)-α-terpineol, with the exception of H579F and H579Y (aromatic substitutions), 100 Abundance which, similar to the wild-type enzyme, generated (−)-limonene as B [%] > 80 the only major product ( 85%). Interestingly, angiosperm MTSs that generate cyclized products carry an aromatic residue (H, F, or 60 Y) in the position corresponding to H579 in (4S)-LS. The for- 40 mation of cyclic alcohols by W324 and H579 mutants provides 20 evidence that these residues are also required for avoiding quenching of the reaction by water, thereby further stabilizing 0 the α-terpinyl cation for subsequent deprotonation. The high- resolution structure of (+)-bornyl diphosphate synthase (at 2.0 Å) indicated the presence of water molecules in the active site of this MTS (19), which could not be discerned in the lower resolution structure (at 2.7 Å) of (4S)-LS (8). (+/−)-Linalool (formed by W324 mutants) would be expected to be generated by water K W D A capture of the linalyl cation, where the charge is closer to the N V F C Y H metal ion-bound pyrophosphate at the top of the active site. (+/−)-α-Terpineol (formed in H579 mutants) would be predicted Fig. 3. Mutant enzymes produce acyclic monoterpene olefins, and acyclic to be produced by reaction with water approaching from the and monocyclic monoterpene alcohols, when amino acid exchanges are bottom of the active site. It is presently unknown whether water made in positions occupied by residues required for stabilizing the α-terpinyl molecules are admitted to the active site in W324 and H579 cation. (A) W324 and (B) H579 mutants. mutants or, if already present in wild-type (4S)-LS, are permitted to react with carbocation intermediates in these mutants.

with L-alanine, the mutant enzymes were mostly unaffected in Which Residues Are Involved in the Final Deprotonation Reaction? their specific activity and fidelity. However, 11 mutants released The ultimate steps of the (4S)-LS reaction cascade are the (−)-limonene as less than 80% of the total monoterpene products. deprotonation of a carbocation and release of the olefin final In these fidelity-impaired enzymes, a residue containing a carbon product. Several authors have previously proposed that the ini- atom within close proximity to a substrate carbon atom was mu- tially released pyrophosphate may act as the catalytic base (3, tated (Fig. 4A). Strong hydrogen bonding below 3 Å can thus 14–17). This is likely correct for bornyl diphosphate synthase occur between donor oxygens or nitrogens of active site amino (BPPS) from culinary sage (Salvia officinalis L.), where the py- acids (e.g., –OH in T349, or S454; –NH– in W324 or H579) and rophosphate is recaptured in the terminating step of the reaction acceptor sites of the substrate or intermediates. By analogy to an (19, 23). However, such a recapture does not occur in other earlier mechanistic proposal for 5-epi-aristolochene synthase characterized MTSs and, if the carbocation was situated and (TEAS) (20), we propose that the carbocation reaction inter- stabilized, as hypothesized, in a hydrophobic pocket of the active mediates of (4S)-LS might move deeper into a hydrophobic site, then the metal ion-bound diphosphate would be too distant pocket [composed of W324, N345, T349, S454, M458, and H579 to act as a base (Fig. 4). Based on quantum mechanical calcu- in (4S)-LS], which would enhance the stabilization of the positive lations, Hong and Tantillo (24) proposed pathways for the for- charge by aromatic residues situated at the bottom of the active mation of BPP by BPPS. In all models, the bicyclic bornyl cation site. The region of positive charge around the divalent metal ions assumes a conformation that positions C2 for a recapture of the interacts directly with the diphosphate leaving group, thereby diphosphate anion, and we propose that, in other MTSs, the immobilizing this anion and preventing a recapture of the allylic α-terpinyl carbocation interacts with the charge-stabilizing aro- carbocation (which would regenerate GPP and terminate the matic residues W324 (conserved) and H579 (or other aromatic reaction) (20, 21). residues in the same position) at the bottom of the active site cavity (separated from the diphosphate anion, which is bound by W324 and H579 Appear to Play Roles in Stabilizing the α-Terpinyl residues forming the top of the active site). Cation. Following the cyclization step, the positive charge of the H579 has the properties of a catalytic base and, in one energy- α-terpinyl cation could be stabilized by cation-π interactions with minimized docking orientation of the α-terpinyl cation, is posi- the electron-rich heteronuclear aromatic rings of W324 and tioned close to the proton to be removed (Fig. 4C). W324 is H579, which would be particularly strong if the carbocation were situated close to the leaving proton in an alternative docking situated, as hypothesized, in the hydrophobic active site pocket orientation of the α-terpinyl cation (Fig. 4D). Whereas H579 (Fig. 4B). Analogous stabilizing roles had been proposed for aro- could be the primary base in wild-type (4S)-LS, it can be ex- matic residues in sesquiterpene synthases (20, 21). The importance changed with other, nonbasic, residues with only minor effects on

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1501203112 Srividya et al. Downloaded by guest on September 23, 2021 enzyme activity. If W324 were to act as a catalytic base (resulting A in a protonated indole with an approximate pKa of −2) (25), the protonation would occur preferentially at Cγ, which is a possi- D356 bility because the α-terpinyl cation is a strong conjugate acid (pKa ∼−10). The significant reduction of specific enzyme ac- D469 tivity in all W324 mutants could be interpreted as evidence that the termination reaction (rather than the initial isomerization) (10) has D352 become rate limiting. The fact that the W324A/H579A double S454 I348 mutant had no measurable enzyme activity is consistent with a L492 function of these residues as potential catalytic bases. An alterna- M458 tive explanation would be that W324 (and even more so W324/ H579 H579) mutations cause an aberrant binding of GPP. This issue can N345 only be resolved with a series of more detailed kinetic evaluations outside the scope of the current study. The work presented here W324 constitutes the most extensive combination of mutagenesis and product analysis in a model MTS, and it can be concluded that very subtle modifications in active site structure can result not only in alteration of termination chemistry, but also in the regiochemistry and stereochemistry of the cyclization reaction itself. The native B S D356 (4 )-LS, an enzyme of very high fidelity, must therefore guide the reaction course very precisely to avoid such digression. Our data are the foundation for future efforts to better understand the as yet D496 unpredictable outcome of reactions catalyzed by MTSs. D352 Materials and Methods L492 I348 Calculating C–C Distances from (4S)-LS Crystal Structure. Coordinates of the T349 carbon atoms of all amino acids were obtained from the published crystal

structure of (4S)-LS (PDB accession identifier 2ONH; complexed with the C10 BIOCHEMISTRY + δ- substrate analog FLPP). A perl script was used to calculate each distance M458 H579 using the following formula: δ- qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð − Þ = ð − Þ2 + ð − Þ2 + ð − Þ2: W324 D C1 C2 x1 x2 y1 y2 z1 z2 N345

Site-Directed Mutagenesis. The spearmint (4S)-LS cDNA, truncated at R58 to eliminate the plastidial targeting sequence, was cloned into the pSBET vector (26) as described before (8). Point mutations were introduced by PCR C using a modified overlap extension strategy as outlined previously (27). A D496 complete list of primers used in these reactions is provided in Dataset S5.PCR amplicons were digested with BamHI and NdeI, gel purified, and ligated into D352 a similarly digested and purified vector. The products of the ligation reactions were transformed into XL-1 blue competent cells and plated onto LB- agar plates containing 50 μg/mL kanamycin. Individual colonies were picked and bacteria grown under selection pressure. Plasmid DNA was then isolated L492 I348 using a commercial kit (GeneJet Mini Prep kit, Fermentas–Thermo Fisher Scientific) and the inserted gene sequence was confirmed by a commercial service provider (Eurofins Genomics). M458 H579 Recombinant Protein Expression and Purification. Vectors containing (4S)-LS H wild-type (R58) and mutant genes were transformed into E. coli BL21 DE3 cells, which were then grown overnight to an OD of ∼1.0 at 37 °C in LB N345 medium containing 50 μg/mL kanamycin. The cultures were induced with W324 0.5 mM isopropyl-1-thio-β-D-galactopyranoside and grown for another 24 h at 16 °C. Cells were harvested by centrifugation at 2,500 × g for 10 min, sus- pended in a cell disruption buffer [50 mM MOPSO, 10 mM DTT, 10% (vol/vol) D D356 glycerol; pH 7.0], and sonicated on ice three times for 15 s each. The supernatant (400 μL) obtained after centrifugation at 15,000 × g was mixed with 100 mg hydroxyapatite [preequilibrated with 10 mM sodium phos- D469 phate (pH 7.0)] in an Eppendorf tube and gently mixed by tube inversion for 1 h at 4 °C. The hydroxyapatite was then allowed to settle by gravity and the L492 D352 supernatant was removed with a Pasteur pipette. Weakly bound proteins were removed by washing with 50 mM MOPSO (pH 7.0). (4S)-LS was eluted M458 I348 T349 green. (A) Residues lining the active site of (4S)-limonene synthase with H a bound substrate analog based on crystal structural data (9). (B) Proposed W324 H579 stabilization of the α-terpinyl cation intermediate by carbocation-π interactions with H579 and/or W324. (C) Orientation of α-terpinyl cation so that N345 H579 can act as a catalytic base. (D) Orientation of α-terpinyl cation so that W324 can act as a catalytic base. Note that the deprotonation reactions in Fig. 4. Possible roles of W324 and H579. The carbon skeletons of the sub- C and D would both result in a product with the correct stereochemistry strate analog (FLPP) and the α-terpinyl cation intermediate are depicted in [(−)-limonene].

Srividya et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 with 100 mM sodium phosphate (pH 7.0). SDS gel electrophoresis indicated consumed. Kinetic parameters were determined by varying substrate a >90% purity of the recombinant enzymes. concentrations while maintaining other reactants at saturation. Kinetic

constants (Km and Kcat) were calculated by nonlinear regression analysis Enzyme Assays. Purified enzyme (200 μg) was reacted with 0.5 mM GPP (Origin 8; OriginLab). (obtained synthetically according to ref. 28) in 400 μL MOPSO buffer (pH 7.0) overlayed with 100 μL hexane for 16 h at 31 °C (gentle agitation). Enzymatic Carbocation Structure and Docking. The molecular geometry of the α-terpinyl reactions were terminated by vigorous mixing of the aqueous and organic cation was optimized by ab initio electronic structure calculation and mini- phases, and phase separation was achieved by placing samples in a freezer mization (using default values for bond length, bond angles, and dihedral − ( 20 °C) for 2 h. The upper hexane layer was removed for analysis by gas angles) with the Gaussian03 program at the HF/6–31G level (Gaussian Inc.). chromatography with flame ionization detection (model 7890, Agilent The optimized geometry output file was converted into the standard format Technologies) under the following conditions: injector at 250 °C, 20:1 split for the Protein Data Bank. Molecular docking of the α-terpinyl cation in the injection mode (1 μL); detector at 250 °C (H flow at 30 mL/min, airflow at 2 (4S)-LS active site was performed with AutoDock Vina (vina.scripps.edu/) 400 mL/min, makeup flow (He) at 25 mL/min); Cyclodex-B chiral column using default settings. (J&W Scientific 112–2532; 30 m × 0.25 mm, 0.25 μm film thickness) operated at 2 mL/min flow rate with He as carrier gas; oven heating program starting at 40 °C with a ramp to 120 °C at 2°/min, a second ramp ACKNOWLEDGMENTS. The authors thank Ms. Lisa Washburn and Ms. Deanna Heidorn for technical assistance. This work was supported by the to 200 °C at 50 °C/min, and a final hold at 200 °C for 2 min. Peaks were Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic identified based on comparisons of retention indices with those of au- Energy Sciences, US Department of Energy Grant DE-FG02-09ER16054 (to thentic standards and verified where necessary by standard GC-MS B.M.L.) for the biochemical characterization of mutant enzymes and the methods (29). For kinetic assays, enzymatic assay times were adjusted to National Institutes of Health Grant GM-31354 (to R.B.C.) for the genera- 2 h to ensure that no more than 20% of the available substrate was tion of site-directed mutants.

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